The invention is in the field of biological products for the treatment and diagnosis of bacterial infections.
Staphylococci are Gram-positive spherical cells, usually arranged in grape-like irregular clusters. Some are members of the normal flora of the skin and mucous membranes of humans, others cause suppuration, abscess formation, a variety of pyogenic infections, and even fatal septicemia. Pathogenic staphylococci often hemolyze blood, coagulate plasma, and produce a variety of extracellular enzymes and toxins. The most common type of food poisoning is caused by a heat-stable staphylococci enterotoxin.
The genus Staphylococcus has at least 30 species. The three main species of clinical importance are Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. Staphylococcus aureus is coagulase-positive, which differentiates it from the other species. S. aureus is a major pathogen for humans. Almost every person has some type of S. aureus infection during a lifetime, ranging in severity from food poisoning or minor skin infections to severe life-threatening infections. The coagulase-negative staphylococci are normal human flora which sometimes cause infection, often associated with implanted devices, especially in very young, old and immunocompromised patients. Approximately 75% of the infections caused by coagulase-negative staphylococci are due to S. epidermidis. Infections due to Staphylococcus warneri, Staphylococcus hominis, and other species are less common. S. saprophyticus is a relatively common cause of urinary tract infections in young women. The staphylococci produce catalase, which differentiates them from the streptococci.
S. aureus colonization of the articular cartilage, of which collagen is a major component, within the joint space appears to be an important factor contributing to the development of septic arthritis. Hematogenously acquired bacterial arthritis remains a serious medical problem. This rapidly progressive and highly destructive joint disease is difficult to eradicate. Typically, less than 50% of the infected patients fail to recover without serious joint damage. S. aureus is the predominant pathogen isolated from adult patients with hematogenous and secondary osteomyelitis.
In hospitalized patients, Staphylococcus bacteria such as S. aureus are a major cause of infection. Initial localized infections of wounds or indwelling medical devices can lead to more serious invasive infections such as septicemia, osteomyelitis, mastitis and endocarditis. In infections associated with medical devices, plastic and metal surfaces become coated with host plasma and matrix proteins such as fibrinogen and fibronectin shortly after implantation. The ability of S. aureus and other staphylococcal bacteria to adhere to these proteins is essential to the initiation of infection. Vascular grafts, intravenous catheters, artificial heart valves, and cardiac assist devices are thrombogenic and prone to bacterial colonization. Of the staphylococcal bacteria, S. aureus is generally the most damaging pathogen of such infections.
A significant increase in S. aureus isolates that exhibit resistance to most of the antibiotics currently available to treat infections has been observed in hospitals throughout the world. The development of penicillin to combat S. aureus was a major advance in infection control and treatment. Unfortunately, penicillin-resistant organisms quickly emerged and the need for new antibiotics was paramount. With the introduction of every new antibiotic, S. aureus has been able to counter with xcex2-lactamases, altered penicillin-binding proteins, and mutated cell membrane proteins allowing the bacterium to persist. Consequently, methicillin-resistant S. aureus (MRSA) and multidrug resistant organisms have emerged and established major footholds in hospitals and nursing homes around the world. (Chambers, H. F., Clin Microbiol Rev, 1:173, 1988; and Mulligan, M. E., et al., Am J Med, 94:313, 1993) Today, almost half of the staphylococcal strains causing nosocomial infections are resistant to all antibiotics except vancomycin, and it appears to be only a matter of time before vancomycin will become ineffective as well.
There is a strong and rapidly growing need for therapeutics to treat infections from staphylococci such as S. aureus which are effective against antibiotic resistant strains of the bacteria. The U.S. National Institutes for Health has recently indicated that this goal is now a national priority.
Bacterial adherence to host tissue occurs when specific microbial surface adhesins termed MSCRAMMs (Microbial Surface Components Recognizing Adhesive Matrix Molecules) specifically recognize and bind to extracellular matrix (ECM) components, such as fibronectin, fibrinogen, collagen, and elastin. Many pathogenic bacteria have been shown to specifically recognize and bind to various components of the ECM in an interaction which appears to represent a host tissue colonization mechanism. This adherence involves a group of bacterial proteins termed MSCRAMMs (Patti, J., et al., Ann Rev Microbiol, 48:585-617, 1994; Patti, J. and Hook, M., Cur Opin Cell Biol., 6:752-758, 1994).
MSCRAMMs on the bacterial cell surface and ligands within the host tissue interact in a lock and key fashion resulting in the adherence of bacteria to the host. Adhesion is often required for bacterial survival and helps bacteria evade host defense mechanisms and antibiotic challenges. Once the bacteria have successfully adhered and colonized host tissues, their physiology is dramatically altered and damaging components such as toxins and enzymes are secreted. Moreover, the adherent bacteria often produce a biofilm and quickly become resistant to the killing effect of most antibiotics.
A bacterium can express MSCRAMMs that recognize a variety of matrix proteins. Ligand-binding sites in MSCRAMMs appear to be defined by relatively short contiguous stretches of amino acid sequences (motifs). Because a similar motif can be found in several different species of bacteria, it appears as though these functional motifs are subjected to interspecies transfer (Patti and Hook, Curr Opin Cell Biol, 6:752-758, 1994). In addition, a single MSCRAMM can sometimes bind several ECM ligands.
Historically, studies on bacterial adherence have focused primarily on Gram-negative bacteria, which express a wide variety of fimbrial adhesive proteins (designated adhesins) on their cell surface (Falkow, S., Cell, 65:1099-1102, 1991). These adhesins recognize specific glycoconjugates exposed on the surface of host cells (particularly epithelial layers). Employing the lectin-like structures in attachment allows the microorganism to efficiently colonize the epithelial surfaces. This provides the bacteria an excellent location for replication and also the opportunity to disseminate to neighboring host tissues. It has been demonstrated that immunization with pilus adhesins can elicit protection against microbial challenge, such as in Hemophilus influenza induced otitis media in a chinchilla model (Sirakova et al., Infect Immun, 62(5):2002-2020, 1994), Moraxella bovis in experimentally induced infectious bovine keratoconjunctivitis (Lepper et al., Vet Microbiol, 45(2-3):129-138, 1995), and E. coli induced diarrhea in rabbits (McQueen et al., Vaccine, 11:201-206, 1993). In most cases, immunization with adhesins leads to the production of immune antibodies that prevent infection by inhibiting bacterial attachment and colonization, as well as enhancing bacterial opsonophagocytosis and antibody-dependent complement-mediated killing.
The use of molecules that mediate the adhesion of pathogenic microbes to host tissue components as vaccine components is emerging as an important step in the development of future vaccines. Because bacterial adherence is the critical first step in the development of most infections, it is an attractive target for the development of novel vaccines. An increased understanding of the interactions between MSCRAMMs and host tissue components at the molecular level coupled with new techniques in recombinant DNA technology have laid the foundation for a new generation of subunit vaccines. Entire or specific domains of MSCRAMMs, either in their native or site-specifically altered forms, can now be produced. Moreover, the ability to mix and match MSCRAMMs from different microorganisms creates the possibility of designing a single vaccine that will protect against multiple bacteria.
Recent clinical trials with a new subunit vaccine against whooping cough, consisting of the purified Bordatella pertussis MSCRAMMs filamentous hemagglutinin and pertactin, in addition to an inactivated pertussis toxin, are a prime example of the success of this type of approach. Several versions of the new acellular vaccine were shown to be safe and more efficacious than the old vaccine that contained whole bacterial cells (Greco et al., N Eng J Med, 334:341-348, 1996; Gustaffson et al., N Eng J Med, 334:349-355, 1996).
Natural immunity to S. aureus infections remains poorly understood. Typically, healthy humans and animals exhibit a high degree of innate resistance to S. aureus infections. Protection is attributed to intact epithelial and mucosal barriers and normal cellular and humoral responses. Titers of antibodies to S. aureus components are elevated after severe infections (Ryding et al., J Med Microbiol, 43(5):328-334, 1995), however to date there is no serological evidence of a correlation between antibody titers and human immunity.
Over the past several decades live, heat-killed, and formalin fixed preparations of S. aureus cells have been tested as vaccines to prevent staphylococcal infections. A multicenter clinical trial was designed to study the effects of a commercial vaccine, consisting of a staphylococcus toxoid and whole killed staphylococci, on the incidence of peritonitis, exit site infection, and S. aureus nasal carriage among continuous peritoneal dialysis patients (Poole-Warren et al., Clin Nephrol., 35:198-206, 1991). Although immunization with the vaccine elicited an increase in the level of specific antibodies to S. aureus, the incidence of peritonitis was unaffected. Similarly, immunization of rabbits with whole cells of S. aureus could not prevent or modify any stage in the development of experimental endocarditis, reduce the incidence of renal abscess, or lower the bacterial load in infected kidneys (Greenberg, D. P., et al., Infect Immun, 55:3030-3034, 1987).
Currently there is no FDA approved vaccine for the prevention of S. aureus infections. However, a S. aureus vaccine (StaphVAX), based on capsular polysaccharide, is currently being developed by NABI (North American Biologicals Inc.). This vaccine consists of type 5 or type 8 capsular polysaccharides conjugated to Pseudomonas aeruginosa exotoxin A (rEPA). The vaccine is designed to induce type-specific opsonic antibodies and enhance opsonophagocytosis (Karakawa et al., Infect Immun, 56:1090-1095, 1988). Using a refined lethal challenge mouse model (Fattom et al., Infect Immun, 61:1023-1032, 1993) it has been shown that intraperitoneal infusion of type 5 capsular polysaccharide specific IgG reduces the mortality of mice inoculated intraperitoneally with S. aureus. The type 5 capsular polysaccharide-rEPA vaccine has also been used to vaccinate seventeen patients with end-stage renal disease (Welch et al., J Amer Soc Nephrol, 7(2):247-253, 1996). Geometric mean (GM) IgG antibody levels to the type 5 conjugate increased between 13 and 17-fold after the first immunization, however no additional increases could be detected after additional injections. Interestingly, the GM IgM levels of the vaccinated patients were significantly lower than control individuals. Supported by the animal studies, the vaccine has recently completed a Phase II trial in continuous ambulatory peritorneal dialysis patients. The clinical trial showed the vaccine to be safe but ineffective in preventing staphylococcal infections (NABI SEC FORM 10-K405, Dec. 31, 1995). Two possible explanations for the inability of Staph VAX to prevent infections related to peritoneal dialysis in vaccinated patients are that the immunogenicity of the vaccine was too low due to suboptimal vaccine dosing or that antibodies in the bloodstream are unable to affect infection in certain anatomic areas, such as the peritoneum.
Gram-positive bacteria related sepsis is on the increase. In fact between one-third and one-half of all cases of sepsis are caused by Gram-positive bacteria, particularly S. aureus and S. epidermidis. In the United States, it can be estimated that over 200,000 patients will develop Gram-positive related sepsis this year.
Using a mouse model (Bremell et al., Infect Immun. 59(8):2615-2623, 1991), it has been clearly demonstrated that active immunization with M55 domain of the Col-binding MSCRAMM can protect mice against sepsis induced death. Mice were immunized subcutaneously with either M55 or a control antigen (bovine serum albumin) and then challenged intravenously with S. aureus. Eighty-three percent (35/42) of the mice immunized with M55 survived compared to only 27% of the BSA immunized mice (12/45). This a compilation of 3 separate studies.
Schennings, et al, demonstrated that immunization with fibronectin binding protein from S. aureus protects against experimental endocarditis in rats (Micro Pathog, 15:227-236, 1993). Rats were immunized with a fusion protein (gal-FnBP) encompassing beta-galactosidase and the domains of fibronectin binding protein from S. aureus responsible for binding to fibronectin. Antibodies against fusion protein gal-FnBP were shown to block the binding of S. aureus to immobilized fibronectin in vitro. Endocarditis in immunized and non-immunized control rats was induced by catheterization via the right carotid artery, resulting in damaged aortic heart valves which became covered by fibrinogen and fibronectin. The catheterized rats were then infected intravenously with 1xc3x9710 5 cells of S. aureus. The number of bacteria associated with aortic valves was determined 11/2 days after the challenge infection and a significant difference in bacterial numbers between immunized and non-immunized groups was then observed.
A mouse mastitis model was used by Mamo, et al., (Vaccine, 12:988-992, 1994) to study the effect of vaccination with fibrinogen binding proteins (especially FnBP-A) and collagen binding protein from S. aureus against challenge infection with S. aureus. The mice vaccinated with fibrinogen binding proteins showed reduced rates of mastitis compared with controls. Gross examination of challenged mammary glands of mice showed that the glands of mice immunized with fibrinogen binding proteins developed mild intramammary infection or had no pathological changes compared with glands from control mice. A significantly reduced number of bacteria could be recovered in the glands from mice immunized with fibrinogen binding proteins as compared with controls. Mamo then found that vaccination with FnBP-A combined with staphylococcal alpha toxoid did not improve the protection (Mamo, et al., Vaccine, 12:988-992, 1994). Next, Mamo, et al., immunized mice with only collagen binding protein, which did not induce protection against the challenge infection with S. aureus. 
Whole killed staphylococci were included in a vaccine study in humans undergoing peritoneal dialysis (Poole-Warren et al., Clin. Nephrol, 35:198-206, 1991). In this clinical trial, a commercially available vaccine of alpha-hemolysin toxoid combined with a suspension of whole killed bacteria) was administered intramuscularly ten times over 12 months, with control patients receiving saline injections. Vaccination elicited significant increases in the levels of antibodies to S. aureus cells in the peritoneal fluid and to alpha-hemolysin in the serum. However, immunization did not reduce the incidences of peritonitis, catheter-related infections or nasal colonization among vaccine recipients. The lack of protective efficacy in this trial were attributed to a suboptimal vaccine formulation.
Secreted proteins have been explored as components of subcellular vaccines. The alpha toxin is among the most potent staphylococcal exotoxins; it has cytolytic activity, induces tissue necrosis and kills laboratory animals. Immunization with formaldehyde-detoxified alpha toxin does not protect animals from systemic or localized infections, although it may reduce the clinical severity of the infections (Ekstedt, R. D., in The Staphylococci, 385-418, 1972).
One study has evaluated the protective efficacy of antibodies to the S. aureus microcapsule in an experimental model of staphylococcal infection (Nemeth, J. and Lee, J. C., Infect. Immun. 63:375-380, 1995). Rats were actively immunized with killed, microencapsulated bacteria or passively immunized with high-titer rabbit antiserum specific for the capsular polysaccharide. Control animals were injected with saline or passively immunized with normal rabbit serum. Protection against catheter-induced endocarditis resulting from intravenous challenge with the same strain was then evaluated. Despite having elevated levels of anticapsular antibodies, the immunized animals were susceptible to staphylococcal endocarditis and immunized and control animals had similar numbers of bacteria in the blood.
As described in the Detailed Description of the Invention hereinbelow, a number of patents and patent applications describe the gene sequences for fibronectin, fibrinogen, collagen, elastin, and MHC II analogous type binding proteins. These patents and patent applications are incorporated by reference in their entirety. These documents teach that the proteins, fragments, or antibodies immunoreactive with those proteins or fragments can be used in vaccinations for the treatment of S. aureus infections. PCT/US97/087210 discloses the vaccination of mice with a combination of a collagen binding protein (M55 fragment), a fibronectin binding peptide (formulin treated FnBP-A (D1-D3)) and a fibrinogen binding peptide (ClfA).
The lack of adequate protection against staphylococcal infection that has been seen to date from the vaccines described above is likely the result of the failure to generate the proper immune response, perhaps along with improper immunization scheduling or an improper immunization route. Additional factors that also contribute to the poor performance of past vaccines can be reflected in the fact that staphylococcal bacteria such as S. aureus have been observed to temporally regulate the expression of most of its virulence factors via regulatory genes loci agr and sar. For example, S. aureus contains two genes that encode cell surface fibrinogen binding proteins, ClfA and ClfB. Interestingly, ClfA is predominately expressed in early exponential growth, while ClfB is expressed later in the growth phase. Accordingly, the antigens that the invading organism presents to the host in vivo may not be the same as those used in the vaccine. In addition, not every S. aureus antigen is expressed on every isolate. For example, only about 50% of S. aureus clinical isolates express the gene cna, which encodes for the collagen binding MSCRAMM. To generate an effective immunotherapeutic against S. aureus, the vaccine must be multi-component and contain antigens that span the growth cycle as well as include antigens that are expressed by a majority of S. aureus isolates.
Despite the advances in the art of compositions for the treatment of infections from staphylococcal bacteria such as S. aureus, there remains a need to provide a more effective product, and preferably one that exhibits a broad spectrum immunization against staphylococcal bacteria of various strains, and to particular proteins which may be expressed at different stages of the bacterial growth phase.
Therefore, it is an object of the invention to provide a new therapeutic composition for immunization against infections from staphylococcal bacteria such as S. aureus and S. epidermidis. 
It is another object of the present invention to provide a vaccine that will provide protection against mastitis, arthritis, endocarditis, septicemia, and osteomyelitis, furunculosis, cellulitis, pyemia, pneumonia, pyoderma, supporation of wounds, food poisoning, bladder infections and other infectious diseases.
It is another object of the present invention to provide a therapeutic composition that immunizes against staphylococcal infection, enhances the amount of intracellular killing of staphylococcal bacteria, and increases the rate of phagocytosis of staphylococcal bacteria.
It is still another object of the present invention to provide a composition that will further protect the host by neutralizing exotoxins.
It has been discovered that the treatment of staphylococcal infections can be significantly enhanced by immunization with certain selected combinations of bacterial binding proteins or fragments thereof, or antibodies to those proteins or fragments. The proteins or fragments can be used in active vaccines, and the antibodies in passive vaccines. Alternatively, the combinations can be used to select donor blood pools for the preparation of purified blood products for passive immunization. By careful selection of the proteins, fragments, or antibodies, a vaccine is provided that imparts protection against a broad spectrum of Staphylococcus bacterial strains and against proteins that are expressed at different stages of the logarithmic growth curve.
The vaccine and products described herein respond to the urgent need of the medical community for a substitute for small molecule antibiotics, which are rapidly losing effectiveness. The vaccines are a significant improvement over the prior art, which while generally teaching the use of MSCRAMMs to impart immunization, did not teach which combinations of the large number of known MSCRAMMs should be used to impart superior protection.
In one embodiment of the invention, a composition is provided that includes at least a collagen binding protein or peptide (or an appropriate site directed mutated sequence thereof) such as CNA, or a protein or fragment with sufficiently high homology thereto, in combination with a fibrinogen binding protein, preferably Clumping factor A (xe2x80x9cClfAxe2x80x9d) or Clumping factor B (xe2x80x9cClfBxe2x80x9d), or a useful fragment thereof or a protein or fragment with sufficiently high homology thereto.
In another embodiment of the invention, a composition is provided that includes at least a fibronectin binding protein or peptide (or an appropriate site directed mutated sequence thereof), or a protein or fragment with sufficiently high homology thereto, in combination with the fibrinogen binding protein, preferably A or B (ClfA or ClfB, respectively), or a useful fragment thereof or a protein or fragment with sufficiently high homology thereto.
In a third embodiment, a composition is provided that includes at least the fibrinogen binding protein A (ClfA) and the fibrinogen binding protein B (ClfB), or useful fragments thereof or a protein or fragment with sufficiently high homology thereto.
In a fourth embodiment, a composition is provided that includes at least a fibronectin binding protein or peptide (or an appropriate site directed mutated sequence thereof), or a protein or fragment with sufficiently high homology thereto, in combination with (i) the fibrinogen binding protein A and B (ClfA and ClfB), or a useful fragment thereof or a protein or fragment with sufficiently high homology thereto; and (ii) a collagen binding protein or useful fragment thereof.
In an additional embodiment, a composition is provided that includes the components of the prior embodiments in combination with an elastin binding protein or peptide or a protein or fragment with sufficiently high homology thereto.
In another embodiment, a composition is provided that includes the components of the prior embodiments in combination with a MHC II analogous protein or peptide or a protein or fragment with sufficiently high homology thereto.
In another embodiment, a composition is provided that includes the components of any of the prior combinations in combination with a bacterial component to increase the rate of phagocytosis of the staphylococcal bacteria. In a one such embodiment, the bacterial component comprises a capsular polysaccharide, such as capsular polysaccharide type 5 or type 8.
In an additional embodiment, a composition is provided that includes any of the prior combinations in combination with the extracellular matrix-binding proteins SdrC, SdrD, SdrE or a consensus or variable sequence amino acid motif, or useful fragments thereof or proteins or fragments with sufficiently high homology thereto.
In an additional embodiment, a composition is provided that includes and of the prior combinations in combination with the extracellular matrix-binding proteins SdrF, SdrG, or SdrH, or a consensus or variable sequence amino acid motif, or useful fragments thereof or proteins or fragments with sufficiently high homology thereto. This embodiment is particularly effective in developing vaccines that can be useful with regard to both coagulase-positive and coagulase-negative staphylococcal bacteria.
In another embodiment, a composition is provided that includes at least the extracellular matrix-binding proteins SdrC, SdrD and SdrE or useful fragments thereof, such as the consensus or variable sequence amino acid motif, or a protein or fragment with sufficiently high homology thereto.
Alternatively, compositions are provided that include monoclonal or polyclonal antibodies which are immunoreactive to the selected combination of described components. These compositions can be used in vaccinations to treat patients infected with Staphylococcus infections.
In other embodiments of the invention, the combinations of proteins, fragments or antibodies as described are used in diagnostic kits.
As described below, proteins and peptides to be used in the composition which bind to fibronectin, fibrinogen, collagen, and elastin are known. Alternatively, one can identify new fibronectin, fibrinogen, collagen, and elastin binding proteins, or the epitopes thereof for use in the composition. Methods of identifying a peptide of a binding domain of a binding protein that binds to the ligand of choice are known. For example, one can contact a candidate protein or peptide with the ligand under conditions effective to allow binding of the ligand to the binding domain of a binding protein, and identify a positive candidate peptide that binds to the ligand.
Antibodies that bind to the binding domains of the composition proteins or peptides can be generated by administering to an animal a pharmaceutical composition comprising an immunologically effective amount of the combination of proteins or peptides, even though the peptide does not specifically bind to the ECM.
The combination of the isolated, recombinant or synthetic MSCRAMM proteins, or active fragments thereof or fusion proteins thereof, are also useful as scientific research tools to identify staphylococcal binding sites on the host ECM molecules, thereby promoting an understanding of the mechanisms of bacterial pathology and the development of antibacterial therapies. Furthermore, the isolated, recombinant or synthetic proteins, or antigenic portions thereof (including epitope-bearing fragments), or fusion proteins thereof can be administered to animals as immunogens or antigens, alone or in combination with an adjuvant, for the production of antisera reactive with MSCRAMM proteins. In addition, the proteins can be used to screen antisera for hyperimmune patients from whom can be derived antibodies having a very high affinity for the proteins. Antibodies isolated from the antisera are useful for the specific detection of staphylococcal bacteria or binding proteins, as research tools, or as therapeutic treatments against staphylococcal infection.
The proteins, or active fragments thereof, and antibodies to the proteins are useful for the treatment of infections from staphylococcal infections from bacteria such as S. aureus as described above; for the development of anti-Staphylococcus vaccines for active or passive immunization; and, when administered as pharmaceutical composition to a wound or used to coat medical devices or polymeric biomaterials in vitro and in vivo, both the proteins and the antibodies are useful as blocking agents to prevent or inhibit the binding of staphylococcal bacteria to the wound site or biomaterials.
Preferably, animal derived antibody is modified so that it is less immunogenic in the patient to whom it is administered. For example, if the patient is a human, the antibody may be xe2x80x9chumanizedxe2x80x9d by transplanting the complimentarily determining regions of the hybridoma-derived antibody into a human monoclonal antibody as described by Jones et al, (Nature 321:522-525 (1986)) or Tempest et al. (Biotechnology 9:266-273 (1991)).
Kits are also provided that are useful as a diagnostic agent for the detection of staphylococcal infections. According to yet another embodiment, anti-MSCRAMM antibodies as well as the MSCRAMM polypeptides of this invention, are useful as diagnostic agents for detecting infection by staphylococcal bacteria, because the polypeptides are capable of binding to antibody molecules produced in animals, including humans that are infected with staphylococcal bacteria such as S. aureus, and the antibodies are capable of binding to particular staphylococcal bacteria or antigens thereof.
Diagnostic agents may be included in a kit which can also include instructions for use and other appropriate reagents. The kit can also contain a means to evaluate the product of the assay, for example, a color chart, or numerical reference chart. The polypeptide or antibody may be labeled with a detection means that allows for the detection of the MSCRAMM polypeptide when it is bound to an antibody, or for the detection of the anti-MSCRAMM polypeptide antibody when it is bound to Staphylococcus bacteria.
The detection means may be a fluorescent labeling agent such as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC), and the like, an enzyme, such as horseradish peroxidase (HRP), glucose oxidase or the like, a radioactive element such as 125I or 51Cr that produces gamma ray emissions, or a radioactive element that emits positrons which produce gamma rays upon encounters with electrons present in the test solution, such as 11C, 15O, or 13N. The linking of the detection means is well known in the art. For instance, monoclonal anti-MSCRAMM polypeptide antibody molecules produced by a hybridoma can be metabolically labeled by incorporation of radioisotope-containing amino acids in the culture medium, or polypeptides may be conjugated or coupled to a detection means through activated functional groups.
The diagnostic kits of the present invention may be used to detect the presence of a quantity of Staphylococcus bacteria or anti-Staphylococcus antibodies in a body fluid sample such as serum, plasma or urine. Thus, in preferred embodiments, an MSCRAMM polypeptide or anti-MSCRAMM polypeptide antibody composition of the present invention is bound to a solid support typically by adsorption from an aqueous medium. Useful solid matrices are well known in the art, and include crosslinked dextran; agarose; polystyrene; polyvinylchloride; cross-linked polyacrylamide; nitrocellulose or nylon-based materials; tubes, plates or the wells of microtiter plates. The polypeptides or antibodies of the present invention may be used as diagnostic agents in solution form or as a substantially dry powder, e.g., in lyophilized form.